Spectroscopic Properties of Amine-substituted Analogues of Firefly Luciferin and Oxyluciferin

Article abstract of DOI:10.1111/php.12654

Spectroscopic and photophysical properties of firefly luciferin and oxyluciferin analogues with an amine substituent (NH₂, NHMe and NMe₂) at the C6' position were studied based on absorption and fluorescence measurements. Their π-electronic properties were investigated by DFT and TD-DFT calculations. These compounds showed fluorescence solvatochromism with good quantum yields. An increase in the electron-donating strength of the substituent led to the bathochromic shift of the fluorescence maximum. The fluorescence maxima of the luciferin analogues and the corresponding oxyluciferin analogues in a solvent were well correlated with each other. Based on the obtained data, the polarity of a luciferase active site was explained. As a result, the maximum wavelength of bioluminescence for a luciferin analogue was readily predicted by measuring the photoluminescence of the luciferin analogue in place of that of the corresponding oxyluciferin analogue.

Full text of DOI:10.1111/php.12654

Received Date : 04-Aug-2016
Revised Date : 26-Aug-2016
Accepted Date : 07-Sep-2016
Article type : Special Issue Research Article
SPECIAL ISSUE RESEARCH ARTICLE
Spectroscopic properties of amine-substituted analogues
of firefly luciferin and oxyluciferin†
Michio Kakiuchi1, Soichiro Ito1, Minoru Yamaji2, Vadim R. Viviani3,4, Shojiro Maki1 and
Takashi Hirano*1
1 Department of Engineering Science, Graduate School of Informatics and Engineering, The
University of Electro-Communications, Chofu, Tokyo 182-8585, Japan
2 Division of Molecular Science, Graduate School of Science and Engineering, Gunma
University, Kiryu, Gunma 376-8515, Japan
3 Department of Physics, Chemistry and Mathematics, Graduate Program of Biotechnology
and Environmental Monitoring, Federal University of São Carlos (UFSCAR), Rodovia João
Leme dos Santos, km 110, Itinga, Sorocaba, SP, Brazil
4 Graduate Program of Evolutive Genetics and Molecular Biology, Federal University of São
Carlos (UFSCAR), São Carlos, SP, Brazil
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/php.12654
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*Corresponding author e-mail: thirano@uec.ac.jp (Takashi Hirano)
†This article is a part of the Special Issue devoted to various aspects of basic and applied research on
Bioluminescence.
ABSTRACT
Spectroscopic and photophysical properties of firefly luciferin and oxyluciferin analogues with an
amine substituent (NH2, NHMe and NMe2) at the C6’ position were studied based on absorption and
fluorescence measurements. Their π-electronic properties were investigated by DFT and TD-DFT
calculations. These compounds showed fluorescence solvatochromism with good quantum yields. An
increase in the electron donating strength of the substituent led to the bathochromic shift of the
fluorescence maximum. The fluorescence maxima of the luciferin analogues and the corresponding
oxyluciferin analogues in a solvent were well correlated to each other. Based on the obtained data, the
polarity of a luciferase active site was explained. As a result, the maximum wavelength of
bioluminescence for a luciferin analogue was readily predicted by measuring the photoluminescence
of the luciferin analogue in place of that of the corresponding oxyluciferin analogue.
INTRODUCTION
Firefly bioluminescence produces photons by a luciferin−luciferase (L−L) reaction, in which firefly
luciferin (LH2) reacts with ATP and O2 under the enzymatic action of luciferase, to give oxyluciferin
(OLH), CO2 and pyrophosphate (PPi) (Schemes 1A and B) (1,2). It is one of the characteristics of
firefly bioluminescence to generate light that varies from green to red colors. The emission colors are
dependent on different varieties of luciferases and conditions surrounding the reaction site, such as
pH, coexisting metal ions, and temperature (3−13). It has been revealed that the light emitter is OLH.
The luminescence properties of OLH and its analogues have been investigated to understand the color
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modulation mechanism (14−34). Breakthrough evidence for the mechanism was reported using a 5,5-
dimethylluciferin adenylate, that is, the light emitter in the excited singlet (S1) state generated by the
L−L reaction has a structure of the keto-phenolate anion (OL−, Scheme 1B) (35,36). Further studies
on the spectroscopic properties of the phenolate anion of 5,5-dimethyl analogue of OLH supported the
proposed mechanism (37). It was clarified that the physical properties of OL− in the S1 state were
modulated both by the strength of the bonding interaction with a counter cation in the luciferase active
site and the polarity of the active site environment.
<Scheme 1>
To confirm the color modulation mechanism, we have been paying attention to aminoluciferin
(1a), which is bioluminescence active and shows red emission by the L−L reaction with Photinus
pyralis (Ppy) luciferase in a pH-independent manner (38). Our previous studies of the 5,5-dimethyl
analogue of 1a (Scheme 1C) led to the conclusion that the S1 state of aminooxyluciferin (2a) in the
keto form is the light emitting center of the L−L reaction of 1a (39,40). Because 2a will mainly exist
as the enol form in solutions like OLH (31), 5,5-dimethyl analogue 3a is useful for predicting the
spectroscopic property of the keto form of 2a (39). Based on the spectroscopic property of 3a, it was
suggested that the amino (NH2) group of 2a works as an electron donating substituent toward the π-
electronic system for the polarized property of the S1 state showing fluorescence solvatochromism.
Because the NH2 group is basic contrast to the acidic hydroxy (OH) group, the physical properties of
the S1 state of 2a will be modulated only by the polarity of the luciferase active site (41). To support
the suggested mechanism, it is required to understand the accurate photophysical property of the keto
form of 2a by using 3a.
Furthermore, a variety of amine-substituted luciferin analogues such as amine-functionalized
analogues (42,43), fluorophore conjugated analogues (44−46), π-modified analogues (47−50) and
cyclic amine-fused analogues (51,52) were recently prepared, and their luminescence properties were
investigated for imaging applications. For these studies, luciferin analogues 1b and 1c with the
methylamino (NHMe) and dimethylamino (NMe2) groups, respectively, were investigated as standard
substrates for the L−L reactions (44,45,51,52). Interestingly, the emission maxima (λbl) of the L−L
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reactions of 1b and 1c with Ppy luciferase are red-shifted compared to that of 1a. While the emitting
site on the L−L reaction of a luciferin analogue was the S1 state of the corresponding oxyluciferin
analogue, the λbl value was evaluated based on the fluorescence property of the luciferin analogue in
place of the oxyluciferin analogue (44, 52) considering the similarity of LH2 and OLH in the
spectroscopic behaviors (16, 53). In fact, it was found that the fluorescence emission maxima (λfl) of
the cyclic amine-fused luciferin analogues are linearly correlated with their λbl values (52). To confirm
the relationship between the λbl and λfl values of a luciferin analogue, we require deep understanding
of the spectroscopic properties of a luciferin analogue and the corresponding oxyluciferin analogue. In
the present work, we investigated the spectroscopic and photophysical properties of luciferin
analogues 1a-c and 5,5-dimethyloxyluciferin analogues 3a-c having the amine substituent. The
Hammett σp values of the NH2, NHMe, and NMe2 groups are −0.66, −0.70, and −0.83, respectively,
indicating the order of the electron donating strength of the substituent is NH2<NHMe<NMe2 (54).
We were, therefore, able to systematically evaluate the spectroscopic and photophysical data for 1a-c
and 3a-c based on the amine substituent effect on the electronic properties of their S1 states. The
results were applicable to analyzing the λbl values of the L−L reactions of 1a-c with Ppy luciferase.
MATERIALS AND METHODS
General. Melting points were determined with a Yamato MP-21 apparatus. IR spectra were obtained
with a Nicolet 6700 spectrometer with an ATR attachment. High-resolution electro-spray ionization
(ESI) mass spectra were recorded on a JEOL JMS-T100LC mass spectrometer. 1H NMR spectra were
obtained with a JEOL ECA-500 instrument (500 MHz). UV/visible absorption spectra were measured
with Agilent Technologies Cary 60 spectrophotometer (scan speed, 600 nm/min; data interval, 1 nm).
Fluorescence spectra and fluorescence quantum yields were measured with a Hamamatsu Photonics
Quantaurus-QY absolute PL quantum yields measurement system. Fluorescence lifetimes (τf) were
measured with a Hamamatsu Photonics Tau time-correlated single-photon counting fluorimeter
system. Spectroscopic measurements were performed with sample solutions (1.0 × 10−5 mol/L) in a
quartz cuvette (1 cm path length) at 25 1 °C. Spectral-grade solvents were used for the
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measurements of UV/visible absorption and fluorescence. Bioluminescence spectra were measured
with an ATTO AB-1850 spectrophotometer (data interval, 1 nm). Density functional theory (DFT)
calculations were performed with the Gaussian 09 program (Rev. D.01) (55). DFT includes the
B3LYP functional with the 6-31+G(d) basis set (56−58). Molecular graphics were made with
GaussView, Version 5 (59).
Preparation of luciferin analogues 1a-c and oxyluciferin analogues 3a-c. Luciferin analogues 1a-c
and oxyluciferin analogues 3a and 3c were prepare according to the literatures (38,39,44,51).
Oxyluciferin analogue 3b was synthesized from 2-cyano-6-methylaminobenzothiazole as followed.
Oxyluciferin analogue 3b. Ethyl 2-(ethoxycarbonothioylthio)-2-methylpropanoate (187 mg, 0.55
mmol) was dissolved in ethylenediamine (2.8 ml) under Ar, and the reaction mixture was stirred at
room temperature for 2 h. The reaction was quenched by adding saturated NH4Cl aqueous solution,
and ethyl 2-mercapto-2-methylpropanoate was extracted with dichloromethane (50 mL × 2). The
organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The yellow oily
residue containing ethyl 2-mercapto-2-methylpropanoate (141 mg) was dissolved in dehydrated
ethanol (4 mL) under Ar, and 2-cyano-6-methyaminobenzothiazole (50 mg, 0.27 mmol) and
triethylamine (120 μL, 8.8 mmol) were added to the solution. After heating at reflux for 3 h, the
cooled reaction mixture was concentrated in vacuo. The residue was purified by silica gel TLC twice
[hexane/chloroform (1:2) and chloroform/ethyl acetate (2:1)], to give 3b (14 mg, 18%) as reddish
brown crystals: mp 257−260 °C. δH (CDCl3) 1.72 (6 H, s), 2.95 (3 H, d, J 4.6 Hz), 4.36 (1 H, br s),
6.86 (1 H, dd, J 2.3 Hz, J 8.9 Hz), 6.97 (1 H, d, J 2.3 Hz), and 7.94 (1 H, d, J 9.2 Hz). ν /cm−1 3384,
2965, 2917, 1712, 1614, and 1552. m/z (ESI) Found: 292.0577 ([M+H]+). C13H14N3OS2 requires
292.0578; Found: 314.0408 ([M+Na]+). C13H13N3NaOS2 requires 314.0398.
RESULTS AND DISCUSSION
Spectroscopic properties of luciferin analogues 1a-c and oxyluciferin analogues 3a-c.
UV/visible absorption and fluorescence spectra of luciferin analogues 1a-c were measured in DMSO,
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acetonitrile, 2-propanol, methanol, and water (Figure 1). Unfortunately, 1a-c showed low solubility in
chloroform and benzene. UV/visible absorption and fluorescence spectra of oxyluciferin analogues
3a-c were measured in p-xylene, benzene, chloroform, DMSO, acetonitrile, 2-propanol, and methanol
(Figure 2). The analogues 3a-c were insoluble and unstable in water. The spectroscopic data of the
absorption maxima (λab) of the lowest energy bands of the absorption and the fluorescence emission
maxima (λfl) for 1a-c and 3a-c are summarized in Tables 1 and 2, respectively. In the case of luciferin
analogues, the ranges of the λfl values in the used solvents are 472−520 nm for 1a, 486−534 nm for
1b, and 500−562 nm for 1c, and their Φf values are over 0.23. In the case of oxyluciferin analogues,
the ranges of the λfl values in the used solvents are 494−607 nm for 3a, 511−623 nm for 3b, and
535−656 nm for 3c, and their Φf values are over 0.16. Interestingly, the λab and λfl values of 1a-c and
3a-c show small red shifts with increasing the electron donating strength of the amine substituent,
indicating that the amine substituents play a role in modulating the energy levels of the frontier
orbitals of 1a-c and 3a-c. The solvent dependent variations of the λfl values of 1a-c and 3a-c are larger
than those of λab values, indicating that the excited singlet (S1) states of both 1a-c and 3a-c have
polarized character more than the corresponding ground (S0) states (39).
<Figure 1>
<Figure 2>
<Table 1>
<Table 2>
The energy values of Efl (in kcal mol−1) estimated from the λfl values of 1a-c and 3a-c were
correlated with the ET(30) values (60) (Figure 3). The data for all compounds in DMSO show a
significant deviation from the linear correlations, suggesting that strong solvating interactions
between the solvent and the excited molecules affect the π-electronic stabilities of 1a-c and 3a-c.
Thus, the correlations between the Efl and ET(30) values were analyzed by linear regression without
the data in DMSO (dotted lines in Figure 3) expressed with equation (1).
Efl = a ET(30) + b
(1)
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From the slope and the intercept of the line, the coefficients (a and b) were determined, respectively,
and summarized in Table 3. The negative correlations (a < 0) indicate that the S1 states of 1a-c and
3a-c have dipole character more than the corresponding S0 states. The electron donating strengths of
the amine substituents of 1a-c and 3a-c clearly determine the Efl values and have an insignificant
effect on the slopes of the linear correlations.
<Table 3>
<Figure 3>
Figure 4A shows the Efl values of 1a-c and 3a-c in acetonitrile plotted as a function of the
Hammett σp values. Since the plots showed straight lines with positive slopes, it is shown that the Efl
values of both 1a-c and 3a-c linearly decrease with an increase of the electron donating strength of the
amine substituent. Therefore, the Efl values of 1a-c and 3a-c in a solvent will have linear correlation.
Figure 4B shows the Efl values of 3a-c in acetonitrile, 2-propanol and methanol plotted as the
functions of the Efl values of 1a-c in the same solvents. The plots showed straight lines as expressed
with equation (2).
Efl(3) = c Efl(1) + d
(2)
From the slope and intercept of the line, the values of c and d were, respectively, determined to be 1.2
and −21 kcal mol−1 for acetonitrile, 1.4 and −35 kcal mol−1 for 2-propanol, and 1.1 and −18 kcal mol−1
for methanol. The obtained c values are similar among these solvents.
<Figure 4>
Fluorescence lifetimes of luciferin analogues 1a-c and oxyluciferin analogues 3a-c.
Fluorescence lifetimes (τf) of 1a-c in acetonitrile and water and 3a-c in benzene and acetonitrile are
listed in Table 4. By using the τf and Φf values, the rate constants (kf) of the fluorescence emission
−1
process were estimated using the equation, kf = Φf τf . The quantum yields (Φnr) and rate constants
(knr) of the nonradiative decay processes were estimated with the equations, Φnr = 1−Φf and knr = Φnr
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−1
τf , respectively. The kf values of 1a-c and 3a-c are similar to each other (ca. 1−2 × 108 s−1), while the
knr values are slightly varied by the difference in the amine substituents and in the solvents. Because
the kf values of 3a-c are slightly greater than the knr values, 3a-c show great Φf values compared to 1a-
c.
<Table 4>
DFT calculations of luciferin analogues 1a-c and oxyluciferin analogues 2a-c and 3a-c.
To analyze the relationship between the spectroscopic and the π-electronic properties of 1a-c and 3a-
c, DFT and TD-DFT calculations of 1−3 were carried out with the B3LYP/6-31+G(d) method (Table
5). The NHMe group at the C6’ position of 1b, 2b and 3b provide two conformations I and II. The
example for 1b is illustrated in Scheme 2. Thus, the data of the conformers I and II for 1b, 2b and 3b
were also shown in Table 5. The heats of formation of I and II for 1b, 2b and 3b are similar to each
other. The data are deposited in ESI as listed in Table S1.
<Table 5>
<Scheme 2>
The S0 S1 transitions of all the compounds are of π,π* for the main contribution of the HOMO
LUMO configurations. The energy differences (ΔEH−L) between HOMO and LUMO and the
wavelengths (λtr) estimated from the calculated transition energies support the observations that the λab
and λfl values of 1a-c and 3a-c are red-shifted with an increase in the electron donating strength of the
amine substituent. The increase of the electron donating strength of the substituent raises both the
energy levels of HOMO and LUMO. In particular, the HOMO levels exhibit significant increases
compared with the LUMO levels, resulting in decreases of the ΔEH−L value.
The calculated ΔEH−L values of 1a-c are greater than those of 3a-c. This result matches the
observation that the λab and λfl values of 1a-c are blue-shifted compared to those of 3a-c. The
difference in the ΔEH−L values is caused by the reason that the 4,5-dihydrothiazole ring of 1a-c have a
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π-electronic conjugation smaller than the 4(5H)-thiazolone ring of 3a-c. To compare the π-electronic
character of 1a-c and 3a-c, electronic distributions of the HOMOs and LUMOs of 1a-c and 3a-c are
deposited in ESI (Figure S3). The HOMO and LUMO energy levels of 1a-c are higher than those of
3a-c. In particular, the difference in the LUMO energy levels of 1a-c and 3a-c is greater than that in
the HOMO energy levels. This difference in the LUMO energy levels is caused by the reason that the
π-electronic distributions in the LUMOs of 3a-c are largely contributed from the thiazolone ring,
whose π-conjugation is larger than the 4,5-dihydrothiazole ring of 1a-c.
The analogues 2a-c and 3a-c show similar values in each term of the calculated data (Table 5),
indicating that the introduction of the two methyl groups at the C5 position of 2a-c has little effect on
the π-electronic character of 2a-c as explained in the previous report on the 5,5-dimethyl analogue of
OLH (37). In fact, the difference in the λtr values of 2a-c and 3a-c are less than 2 nm, supporting that
the 5,5-dimethyl analogues 3a-c are useful compounds for predicting the spectroscopic properties of
the keto form of 2a-c.
For the NHMe analogues 1b, 2b, and 3b, the difference in the π-electronic character of the
conformers I and II were investigated. We regard the differences in the ΔEH−L and λtr values between
I and II as being small. For instance, the conformers I and II of 1b have the λtr values being 368 and
373 nm, respectively.
Bioluminescence properties of luciferin analogues 1a-c.
Bioluminescence properties of 1a-c with Ppy luciferase have been reported by several groups,
showing that the emission maxima (λbl) of the L−L reactions of 1a-c are red-shifted with increasing
the electron donating ability of the amine substituent (44,45,51,52). We reinvestigated the L−L
reactions of 1a-c with Ppy luciferase in GTA buffer at pH 8.0 and 6.0, and obtained the reproduced
result deposited in ESI (Figure S4). The λbl values are 595, 610, and 620 nm for 1a, 1b, and 1c,
respectively, at pH 8.0 and 6.0. Because the excited light emitters generated by the reactions of 1a-c
are the keto form of 2a-c (39,40), the λbl values of 1a-c are reasonably evaluated by the λfl values of
3a-c in place of 2a-c. The λbl values are similar to the λfl values of 3a in 2-propanol (593 nm), 3b in 2-
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propanol (604 nm), and 3c in acetonitrile (621 nm), respectively. This result indicates that the S1
states of 2a-c generated from 1a-c are stabilized by the microenvironments of the luciferase active site
as they are by the solvation in 2-propanol and acetonitrile. Thus, the Efl−ET (30) plots in Figure 3 are
useful as rulers for evaluating the polarity of a luciferase active site as proposed in the previous
reports (39−41).
Figure 5 shows the energy values of Ebl (in kcal mol−1) estimated from the λbl values on the L−L
reactions of 1a-c plotted as a function of the Efl value for 1a-c and 3a-c in acetonitrile. The plots
showed straight lines as expressed with equation (3).
Ebl = e Efl(1) + f
(3)
From the slope and intercept of the line, the values of e and f were, respectively, determined to be
0.52 and 17 kcal mol−1 for 1a-c, and 0.43 and 26 kcal mol−1 for 3a-c. Because the Efl values of 1a-c
and 3a-c in a solvent are linearly correlated to each other (Figure 4B), it is reasonable for the Ebl−Efl
plots in Figure 5 to show good linear correlations. This result indicates that the S1 states of 2a-c
generated from 1a-c locate at the similar position in the active site of Ppy luciferase, whose
microenvironments provide a polarity equally stabilizing the π-electron systems of the excited
molecules. Similar result was obtained for the cyclic amine-fused luciferin analogues (52). The results
obtained in this work are adoptable for predicting the λbl value of bioluminescence from a luciferin
analogue with the λfl of the photoluminescence in a particular solvent based on the similarity in the
spectroscopic behaviors of luciferin analogues and the corresponding oxyluciferin analogues.
<Figure 5>
CONCLUSION
We investigated the spectroscopic properties of luciferin analogues 1a-c and oxyluciferin analogues
3a-c having the amine substituent (NH2, NHMe and NMe2) with the different electron donating
strength. Their photophysical properties were analyzed by fluorescence lifetime measurements. All of
1a-c and 3a-c show fluorescence solvatochromism with appreciably large Φf values. The fluorescence
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solvatochromism is originated from strongly polarized character of the S1 state. The increase in the
electron donating strength of the amine substituent for 1a-c and 3a-c leads to the bathochromic shift
in the λfl value. The Efl values estimated from the λfl values of 1a-c and 3a-c are linearly correlated to
the Hammett σp values. In addition, it was confirmed that the Efl values of 1a-c are linearly correlated
to those of 3a-c. The substituent effect of the amine family on the π-electronic properties of the
luciferin and oxyluciferin skeletons were reasonably explained by the DFT and TD-DFT calculation
data. Based on the obtained spectroscopic data of 1a-c and 3a-c, we confirmed that the Ebl values
estimated from the λbl values on the L−L reactions of 1a-c with Ppy luciferase are shown to be
expressed with equation (3) as a function of the Efl value for 1a-c and 3a-c (Figure 5). The result
supports the usefulness of the method to predict the λbl values of luciferin analogues with the λfl values
of the luciferin analogues themselves in a particular solvent, when the S1 states of oxyluciferin
analogues produced by the L−L reactions locate at a similar position in the luciferase active site. The
conclusion of this study provides a theoretical guarantee to design a novel luciferin analogue for a
desired purpose based on the fluorescence property of the luciferin analogue in place of the
corresponding oxyluciferin analogue.
Acknowledgements − We thank the Information Technology Center of the University of Electro-
Communications for technical assistance in computing the quantum chemical calculations.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Figure S1. Fluorescence decay curves of 1a (a and b), 1b (c and d) and 1c (e and f) in acetonitrile and
water at 295 K.
Figure S2. Fluorescence decay curves of 3a (a and b), 3b (c and d) and 3c (e and f) in benzene and
acetonitrile 295 K.
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Figure S3. HOMOs and LUMOs of 1a-c and 3a-c. The data of 1b and 3b are for the conformers I.
Figure S4. Bioluminescence spectra for the L−L reactions of 1a-c with Ppy luciferase at (A) pH 8.0
and (B) pH 6.0 at room temperature.
Table S1. Heats of formation of 1−3 with DFT using B3LYP/6-31+G(d).
Table S2. Cartesian Coordinates (in Å) of luciferin analogue 1a (R = NH2).
Table S3. Cartesian Coordinates (in Å) of luciferin analogue 1b-I (R = NHMe).
Table S4. Cartesian Coordinates (in Å) of luciferin analogue 1b-II (R = NHMe).
Table S5. Cartesian Coordinates (in Å) of luciferin analogue 1c (R = NMe2).
Table S6. Cartesian Coordinates (in Å) of oxyluciferin analogue 2b-I (R = NHMe).
Table S7. Cartesian Coordinates (in Å) of oxyluciferin analogue 2b-II (R = NHMe).
Table S8. Cartesian Coordinates (in Å) of oxyluciferin analogue 3b-I (R = NHMe).
Table S9. Cartesian Coordinates (in Å) of oxyluciferin analogue 3b-II (R = NHMe).
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Tables
Table 1. Electronic absorption properties of luciferin analogues 1a-c and oxyluciferin analogues 3a-c
in various solvents at 298 K.
Solvent [ET(30)a]
λab / nm (ε / 104 dm3 mol−1 cm−1)b
1a (NH2)
---
1b (NHMe) 1c (NMe2)
3a (NH2)
415 (1.8)
417 (1.7)
423 (1.5)
470 (2.2)
434 (2.0)
467 (1.9)
455 (1.9)
---
3b (NHMe) 3c (NMe2)
p-Xylene [33.1]
Benzene [34.3]
Chloroform [39.1]
DMSO [45.1]
---
---
443 (1.9)
446 (2.0)
455 (1.8)
483 (2.0)
458 (2.0)
479 (2.1)
473 (2.0)
---
465 (2.6)
469 (2.6)
483 (2.7)
492 (2.4)
476 (2.5)
480 (2.5)
486 (2.5)
---
---
---
---
---
---
---
375 (1.6)
359 (1.3)
360 (1.3)
362 (1.4)
351 (1.4)
386 (1.3)
378 (1.1)
381 (1.3)
382 (1.2)
373 (0.98)
388 (1.8)
386 (1.5)
379 (1.7)
387 (1.7)
383 (1.2)
Acetonitrile [45.6]
2-Propanol [48.4]
Methanol [55.4]
Water [63.1]
a ET(30) in kcal mol−1. b Absorption maximum (λab) and extinction coefficient (ε) in parenthesis.
Table 2. Fluorescence properties of luciferin analogues 1a-c and oxyluciferin analogues 3a-c in
various solvents at 298 K.
λfl / nm (Φf)b
Solvent [ET(30)a]
1a (NH2)
---
1b (NHMe) 1c (NMe2)
2a (NH2)
494 (0.44)
495 (0.54)
511 (0.85)
593 (0.79)
567 (0.84)
593 (0.69)
607 (0.42)
---
2b (NHMe) 2c (NMe2)
p-Xylene [33.1]
Benzene [34.3]
Chloroform [39.1]
DMSO [45.1]
---
---
511 (0.81)
517 (0.85)
534 (0.90)
611 (0.74)
587 (0.81)
604 (0.67)
623 (0.39)
---
535 (0.85)
541 (0.87)
567 (0.90)
640 (0.39)
621 (0.50)
634 (0.46)
656 (0.16)
---
---
---
---
---
---
---
498 (0.49)
472 (0.33)
481 (0.54)
491 (0.58)
520 (0.57)
513 (0.64)
486 (0.50)
489 (0.63)
503 (0.67)
534 (0.57)
514 (0.71)
503 (0.57)
500 (0.70)
519 (0.67)
562 (0.23)
Acetonitrile [45.6]
2-Propanol [48.4]
Methanol [55.4]
Water [63.1]
a ET(30) in kcal mol−1. b Fluorescence maximum wavelengths (λfl) and quantum yields (Φf) in parenthesis.
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Table 3. Coefficients (a and b) for equation (1).
Compounds
1a (NH2)
a
b / kcal mol−1
−0.32
−0.31
−0.36
−0.54
−0.48
−0.46
76
74
74
76
72
68
1b (NHMe)
1c (NMe2)
2a (NH2)
2b (NHMe)
2c (NMe2)
Table 4. Photophysical properties of 1a-c and 3a-c at 295 K.
kf /108 s−1
a
Φnr
knrc / 108 s−1
b
Compound
Solvent
Φf
τf / ns
( b i
1a (NH2)
)
acetonitrile
H2O
0.33
0.57
0.5
3.8
4.8
3.9
4.5
4.6
1.9
2.3
4.6
3.5
4.8
3.9
3.3
0.87
1.2
1.3
1.3
1.2
1.2
1.8
1.8
2.4
1.7
2.2
1.5
0.67
0.43
0.5
1.8
0.9
1b (NHMe)
1c (NMe2)
3a (NH2)
acetonitrile
H2O
1.3
0.57
0.57
0.23
0.41
0.84
0.85
0.81
0.87
0.5
0.43
0.43
0.77
0.59
0.16
0.15
0.19
0.13
0.5
0.96
0.93
4.1
acetonitrile
H2O
benzene
acetonitrile
benzene
acetonitrile
benzene
acetonitrile
2.6
0.35
0.43
0.4
3b (NHMe)
3c (NMe2)
0.33
1.5
a Determined by kf = Φf τf−1. b Determined by Φnr = 1−Φf. c Determined by knr = Φnr τf−1.
Table 5 Calculation data of 1−3 with DFT and TD-DFT using B3LYP/6-31+G(d).
a
c
Compound
HOMO
−5.91
LUMO
−2.12
ΔEH−L
Transitions b
λtr / nm (f)
Configuration d,e
1a (NH2)
3.80
S0 → S1
354 (0.46)
H → L (0.68)
1b-I (NHMe)
1b-II (NHMe)
−5.69
−5.66
−2.04
−2.06
3.65
3.61
S0 → S1
S0 → S1
368 (0.50)
373 (0.49)
H → L (0.69)
H → L (0.69)
H 1
L ( 0 10)
1c (NMe2)
2a (NH2)
−5.54
−6.19
−2.02
−2.87
3.52
3.31
S0 → S1
S0 → S1
383 (0.51)
403 (0.46)
H → L (0.70)
H → L (0.69)
H 1
L ( 0 12)
2b-I (NHMe)
2b-II (NHMe)
−5.95
−5.92
−2.78
−2.80
3.16
3.11
S0 → S1
S0 → S1
419 (0.53)
428 (0.49)
H → L (0.70)
H → L (0.70)
H 1
L ( 0 10)
2c (NMe2)
−5.80
−2.76
3.03
S0 → S1
439 (0.53)
H → L (0.70)
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3af (NH2)
−6.13
−2.80
3.33
S0 → S1
401 (0.49)
H → L (0.69)
H 1
L ( 0 12)
3b-I (NHMe)
3b-II (NHMe)
−5.90
−5.87
−2.71
−2.73
3.19
3.14
S0 → S1
S0 → S1
418 (0.55)
426 (0.52)
H → L (0.70)
H → L (0.70)
3cf (NMe2)
−5.75
−2.69
3.06
S0 → S1
438 (0.55)
H → L (0.70)
H 1
L ( 0 10)
a Energy difference between HOMO and LUMO. b The π,π* transition to the excited singlet state with the lowest excitation
energy. c Wavelengths estimated from transition energies. d Configuration of excitation. e H, H−1, and L denote the HOMO,
HOMO−1, and LUMO, respectively. f Ref. 39.
Figure Captions
Scheme 1. (A) The luciferin−luciferase (L−L) reaction of firefly bioluminescence. (B) The molecular
structures of luciferin (LH2), oxyluciferin (OLH) and keto-phenolate anion of OLH (OL−). (C)
Amine-substituted luciferin analogues 1 and oxyluciferin analogues 2 and 3.
Scheme 2. Conformers I and II of luciferin analogue 1b.
Figure 1. UV/visible absorption (Abs) and fluorescence (Fl) spectra of 1a (A), 1b (B), and 1c (C) in
DMSO (a), acetonitrile (b), 2-propanol (c), methanol (d), and H2O (e) at 298 K.
Figure 2. UV/visible absorption (Abs) and fluorescence (Fl) spectra of 3a (A), 3b (B), and 3c (C) in
p-xylene (a), benzene (b), chloroform (c), DMSO (d), acetonitrile (e), 2-propanol (f), and methanol
(g) at 298 K.
Figure 3. Efl values of 1a (●), 1b (○), 1c ( ), 3a (■), 3b (□), and 3c ( ) plotted as a function of ET(30).
The data in DMSO are marked with an asterisk.
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Figure 4. (A) Efl values of 1a-c (■) and 3a-c (□) in acetonitrile plotted as a function of the Hammett
σp values. (B) Efl values (Efl(3)) of 3a, 3b, and 3c plotted as a function of Efl values (Efl(1)) of 1a, 1b,
and 1c in acetonitrile (■), 2-propanol ( ) and methanol (□).
Figure 5. Ebl values of 1a-c plotted as functions of Efl values of 1a-c (■) and 3a-c (□) in acetonitrile.
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